Exome sequencing for diagnosis of congenital hemolytic anemia

doi:10.21203/rs.2.24438/v1

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Exome sequencing for diagnosis of congenital hemolytic anemia

https://doi.org/10.21203/rs.2.24438/v1

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published 08 Jul, 2020

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Background: Congenital hemolytic anemia constitutes a heterogeneous group of rare genetic disorders of red blood cells. Diagnosis is based on clinical data, family history and phenotypic testing, genetic analyses being usually performed as a late step. In this study, we explored 40 patients with congenital hemolytic anemia by whole exome sequencing: 20 patients with hereditary spherocytosis and 20 patients with unexplained hemolysis.

Results: A probable genetic cause of disease was identified in 82.5% of the patients (33/40): 100% of those with suspected hereditary spherocytosis (20/20) and 65% of those with unexplained hemolysis (13/20). We found that several patients carried genetic variations in more than one gene (3/20 in the hereditary spherocytosis group, 6/13 fully elucidated patients in the unexplained hemolysis group), giving a more accurate picture of the genetic complexity of congenital hemolytic anemia. In addition, whole exome sequencing allowed us to identify genetic variants in non-congenital hemolytic anemia genes that explained part of the phenotype in 3 patients.

Conclusion: The rapid development of next generation sequencing has rendered the genetic study of these diseases much easier and cheaper. Whole exome sequencing use for congenital hemolytic anemia could provide a more precise and quicker diagnosis, improve patients’ healthcare and probably has to be democratized notably for complex cases.

Internal Medicine

Hematology

hemolysis

red blood cell

membrane

NGS

anemia

congenital

mutation

Congenital hemolytic anemia (CHA) is a group of rare genetic disorders characterized by increased destruction of red blood cells (RBC) [1]. They result from corpuscular causes such as hemoglobin disorders, membrane diseases, RBC enzyme deficiencies or congenital dyserythropoietic anemia (CDA), or from extra-corpuscular causes such as atypical hemolytic and uremic syndromes (aHUS) due to complement pathways dysfunctions.

Membrane disorders[2-5] include hereditary spherocytosis (HS, the most frequent cause in European population with nearly 1/2,000 subjects affected) [6]; hereditary elliptocytosis (HE, from 1/5,000-1/10,000 in European population to 1/100 in West African populations) [7]; hereditary pyropoïkilocytosis (HPP), which is a rare and severe subtype of HE, and hereditary stomatocytosis, with a dehydrated form (incidence of 1/50,000 individuals) and an overhydrated form (incidence lower than 1/100,000) [8]. Transmission is often autosomal dominant (AD) but de novo mutations or autosomal recessive (AR) forms (as is the case in HPP) can occur.

Defaults in enzymatic pathways of RBC metabolism are numerous [9] the most frequent being G6PD and pyruvate kinase deficiencies [10]. Most of them have AR inheritance but some are X-Linked (XL) (as is the case for G6PD deficiency).

CDA are characterized by ineffective central erythropoiesis with progressive secondary iron overload and have AR, AD or XL transmission [11,12]. Peripheral hemolysis can occur and misdiagnosis is often due to phenotypic overlap with other types of CHA [13,14].Clinical features of CHA vary from severe neonatal or even prenatal anemia with high morbidity and transfusion dependence to well-compensated hemolysis without anemia. In addition, clinical manifestations may be increased by associated comorbidities (such as hemochromatosis or hemoglobin variant) or during acute diseases, inflammatory conditions, or pregnancy.

Mutations causing CHA have been identified in more than 100 different genes, some of which are very large. Next generation sequencing (NGS) allows massive parallel sequencing of numerous genes and therefore appears as a very suitable approach for genetic dissection of CHA. Different NGS strategies have been developed over the last few years for CHA (Additional file 1, Supplementary Table S1). They included targeted gene panels (comprising 28 to 76 genes) with a success rate for diagnosis varying from 38% to 90%, depending on probands numbers (ranging from 2 to 62) and subtypes of anemia [15-23], and whole-exome sequencing (WES) in a few studies (limited to 1 to 7 probands, except two studies focusing on 38 Chinese cases of HS [24] and 24 cases of autosomal recessive HS [25]), with success rates between 29 and 100% [14, 24-32]. We used WES to explore 40 CHA patients: 20 with suspected HS and 20 with unexplained hemolysis (UH) despite available phenotype exploration. We first analyzed genomic data using a predefined list of 71 CHA genes (Additional file 1, Supplementary Table 2) and extended the analysis to the entire exome when necessary. This approach allowed us to find a probable molecular cause in 82.5% of patients.

(See Supplementary material for more details).

DNA extraction

Peripheral blood EDTA sample was obtained from patients after written informed consent for genetic analysis. Genomic DNA was extracted using the Flexigene extraction kit, following manufacturer’s recommendations (Qiagen; Hilden, Germany).

Sequencing and data analysis

Library preparation was performed from 100 ng of enzymatically fragmented genomic DNA using the Kapa library hyperprep kit, following manufacturer’s recommendations (KAPABIOSYSTEMS, Wilmington, Massachusetts). Enrichment for exonic sequences was performed using the Medexome kit, following manufacturer’s recommendations (NimbleGen, Madison, Wisconsin).

NGS Sequencing. Captured libraries were sequenced on a Nextseq500 instrument (Illumina, San Diego, California, USA) with high-output FlowCell and reagents, in order to obtain 150 bp paired-end reads.

Bioinformatic analyses. Demultiplexing and .fastq files generation were performed using Bcl2fastq software (v.1.8.4). Alignment and variant calling were performed in duplicate, using an in-house pipeline following the BWA/GATK gold standard best practices and the Seqnext software (JSI, Ettenheim, Germany). The variant calling files (VCF) were annotated using both Annovar and Ingenuity Variant Analysis (Qiagen, Hilden, Germany) on hg19 reference genome. The first-line analysis was focused on 71 known CHA genes (Additionnal file 1, Supplementary Table 2). An extended whole-exome analysis was performed as a second-line analysis in unsolved cases, using IVA software filters (phenotypical ranking tools, biological context). Alamut (Interactive biosoftware, Rouen, France) and IGV (Broad institute, Cambridge, MA 02142, USA) were used as reads viewers. Mean depth of coverage was 175x and depth (>30x) was checked using bam files and alamut on regions of interest. Coverage was correct on our gene list excepted GPX1 and PGD first exon, which could be covered by Sanger sequencing. Exon 1 of PIEZO1 was covered under 30x (15x) and was not analyzed by Sanger sequencing.

In silico pathogenic prediction was performed on novel missense variations using Polyphen-2 (http://genetics.bwh.harvard.edu/pph2/), SIFT (http://sift.jcvi.org/), Mutation Taster (http://www.mutationtaster.org/), align GVGD (http://agvgd.iarc.fr/). MaxEntScan (http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html) and Human Splicing Finder3.0 (http://www.umd.be/HSF/) were used for splicing effect predictions. The CADD score was determined through an online tool (https://cadd.gs.washington.edu/). Protein domain functions were determined by using the SMART software (http://smart.embl-heidelberg.de/) and are available in table S3.

Online genomic variant databases. The public variation database gnomAD (Broad institute, Cambridge, MA 02142, USA, http://gnomad.broadinstitute.org) was checked for presence and allelic frequency of each genetic variation identified in the patients. We filtered variants according to their frequency in gnomAD. For recessive disorders we only kept variants under 5% of minor allele frequency (MAF) and for autosomal dominant ones those under 1% of MAF.

All new variants have been registered in the Clinvar database (https://www-ncbi-nlm-nih-gov.gate2.inist.fr/clinvar/ see Additional file 1, Supplementary table S4)

Variant analysis and interpretation: Variants with MAF up to 5% were excluded and functional classification was done following ACMG criteria [33]. Biochemical and hematologic tests. Standard laboratory tests included cell blood count, blood smear examination, hemolysis markers, RBC density (measured by the phthalate density-distribution method, as described by Bartolucci et al in 2012[34], with density curves profiles), hemoglobin study (beginning with isoelectric focus ligand HPLC. In case of abnormalities, acid agar gel electrophoresis, Itano test, capillary electrophoresis and reverse phase HPLC could be performed), RBC enzymes activity (G6PD, Pyruvate Kinase, Hexokinase routinely measured) .

EMA test (performed following the methods described by King et al in 2000 [35] and Girodon et al in 2008 [36]).

When possible, osmolar gradient ektacytometry (Technicon®) and membrane protein electrophoresis (MPE) were performed.

Sanger sequencing. Sanger sequencing was used to confirm each potentially deleterious variation found by NGS. Primers sequences and PCR conditions are available upon request.

RNA extraction and transcript analyses Total RNA was extracted from blood drawn into Paxgene tubes, using Maxwell 16 extraction technology (Promega, Madison, Wisconsin, USA) following manufacturer recommendations. cDNA was generated, PCR-amplified and sequenced by the Sanger method. Detailed conditions are available upon request.

Patients

Forty patients suffering from non-immune hemolysis with negative direct antiglobulin tests were submitted to exome sequencing. Clinical and biological data are available in Supplementary Tables S5 and S6 (normal values in Additional file 1, Supplementary Table S7). Blood smear results and genetic results are available in tables S8 and S11. In silico analysis of genetic variants is available in Additional file 1, supplementary table S3.

Twenty patients had a HS phenotype, among which 4 were apparently sporadic cases (P1, P3, P13, P14). The 20 other patients had UH, with either discordant or non-conclusive phenotypical tests. For several patients, EMA test results were discordant with ektacytometry. In the other cases, EMA test and ektacytometry were either normal or atypical. Three of these patients had UH associated to a sickle cell trait (A/S).

Genetic results are available in tables 1 and 2 and in Additional file 1, supplementary tables S3 and S8 to S11.Clinico-biological data are available in Additional file 1, supplementary tables S5 to S7.

We defined two groups: one with clear phenotype (HS group) and one with unclear phenotype (UH group). The different types of variants identified are summarized in figure 1.

HS GROUP

We identified the probable genetic cause of hemolysis in all patients with HS. Nineteen patients were heterozygous carriers of one mutation in ANK1 (8 patients), SLC4A1 (6 patients), or SPTB (5 patients). All patients had different mutations, among which 13 were novel and 8 had already been reported. In one family (P11 and affected relatives) with typical HS, patients harbored 2 heterozygous probably damaging variations, one in SPTB (stop-gained) and the other in SPTA1 (already-reported missense mutation found in one case of HPP which also harbored the alpha-Lely polymorphism in trans). Such an association has rarely been reported in the literature [15, 16, 37, 38] In 3 families (P4, P10 and P11), genetic tests could be extended to other affected family members and showed cosegregation of mutations and disease (Figure 2). Three HS patients also had additional clinically significant mutations in non-HS genes. P1 carried a heterozygous mutation of alpha-globin gene, called Tunis-Bizerte hemoglobin, responsible for an alpha- thalassemia trait [39]. P17 carried a variant of uncertain significance (VUS) in PIEZO1 (p.V860M). P2 harbored 2 variants in HFE (H63D and C282Y) initially found during iron overload exploration. In total, 3 patients on 20 in HS group have variations in two different CHA genes.

Table 1: Genetic results of HS patient

Family ID	Gene name	transcript	Nucleotide change	AA change	zygosity	references	gnomAD allele frequency	Polyphen-2	Mutation taster	MaxEnt Scan	CADD score	ACMG class
P1	ANK1	NM_020476	c.5152C>T	p.Gln1718*	het	No	0	NA	NA		37	LP
	HBA1	NM_0005558	c.389T>C	p.L130P	het	Darbellay et al 1995	0	PD	DC		23.4	LP
P2	ANK1	NM_020476	c.1702-2A>C		het	No ref	0	NA	NA	-100%	34	LP
	HFE	NM_000410	c.187C>G	p.H63D	het	Kaczorowska-Hac et al 2016	10.83%	B	P		12.8	VUS
	HFE	NM_000410	c.845G>A	p.C282Y	het	Kaczorowska-Hac et al 2016	3.37%	PD	DC		25.2	P
P3	SLC4A1	NM_000342	c.1458C>G	p.Y486*	het	No	0	NA	NA		35	LP
P4	SLC4A1	NM_000342	c.486-2A>G		het	no	0	NA	NA	-100%	25.3	P
P4-1	SLC4A1	NM_000342	c.486-2A>G		het	no	0	NA	NA	-100%	25.3	P
P5	SPTB	NM_001024858	c.1331_1338del	p.Leu444Profs*3	het	Dhermy et al 1998	0	NA	DC			P
P6	ANK1	NM_020476	c.5497C>T	p.R1833*	het	Hayette et al 1998	0				47	P
P7	SLC4A1	NM_000342	c.1322T>G	p.L441R	het	No	0	PD	DC	No effect	26.8	VUS
P8	ANK1	NM_020476	c.1801-17G>A		het	Duru et al	0			Creation of a cryptic acceptor	8.2	LP
P9	ANK1	NM_020476	c.4462C>T	p.R1488*	het	Ozcan et al 2003	0				36	P
*P10*	ANK1	NM_020476	c.1A>G	p.?	het	no	0	NA	NA	NA	14.3	P
*P10-1*	ANK1	NM_020476	c.1A>G	p.?	het
*P11*	SPTB	NM_001024858	c.2863C>T	p.R955*	het	no	0	NA	NA	NA	37	P
	SPTA1	NM_003126	c.6421C>T	p.R2141W	het	NIss et al 2016	0.2%	PD	DC		27.4	LP
*P11-1*	SPTB	NM_001024858	c.2863C>T	p.R955*	het							P
	SPTA1	NM_003126	c.6421C>T	p.R2141W	het							LP
	SPTA1	NM_003126	c.6531-12C>T		het
*P11-2*	SPTB	NM_001024858	c.2863C>T	p.R955*	het							P
	SPTA1	NM_003126	c.6421C>T	p.R2141W	het							LP
	SPTA1	NM_003126	c.6531-12C>T		het
*P12*	SPTB	NM_001024858	c.4973+5G>A		het	no	0	NA	NA	-100%	16.48	VUS
*P13*	ANK1	NM_020476	c.534delC	p.H178Qfs*75	het	no	0	NA	NA			LP
*P14*	SPTB	NM_001024858	c.5623C>T	p.Q1875*	het	no	0	NA	NA		48	LP
*P15*	SLC4A1	NM_000342	c.1462G>A	p.V488M	het	Alloisio et al 1997	0.00041%	PD	DC	No effect	24.5	P
*P16*	ANK1	NM_020476	c.712-2A>G		het	no	0	NA	NA	-100%	34	LP
*P17*	SLC4A1	NM_000342	c.2423G>A	p.R808H	het	Bogardus et al 2012	0	PD	DC	No effect	33	LP
	PIEZO1		c.2578G>A	p.V860M	het		0.0028%	PossD	DC	No effect		VUS
*P18*	SLC4A1	NM_000342	c.2279G>A	p.R760Q	het	Jarolim et al 1995	0	PD	DC	No effect	29.6	LP
*P19*	SPTB	NM_001024858	c.3436dup	p.L1146Pfs*36	het	no	0	NA	NA			LP
	SPTB	NM_001024858	c.6101G>A	p.S2034N	het	no	0.00041%	B	DC	No effect	22.9	VUS
*P20*	SPTB	NM_001024858	c.3916C>T	p.R1306*	het	no	0	NA	NA		38	LP

Variants description and classification according to ACMG guidelines as benign likely benign (LB) variant of uncertain significance (VUS) likely pathogenic (LP) or pathogenic (P). In silico study of missense variations was assessed thanks to Polyphen-2, Mutation taster and CADD score algorithm. HGMD professional and pubmed web interface were used to check for variants description in litterature. Abbreviations: het: heterozygous state; hom: homozygous state; hem: hemizygous state; F: female; M: male; HS: hereditary spherocytosis; gnomAD: genome agregation database https://gnomad.broadinstitute.org ; ND: not done; NA: not applicable; DC: disease causing; P: polymorphism; PD: probably damaging; PossD: possibly damaging; B: benign

Table 2: Genetic results of UH patients

Family ID	Gene name	Transcript	Nucleotide change	AA change	zygosity	references	gnomAD allele frequency	Polyphen-2	Mutation taster	MaxEnt Scan	CADD score	ACMG class
P21	SPTA1	NM_003126	c.6600+5G>T		het	no	0	NA	NA	-62.5%	18.74	VUS
	SPTA1	NM_003126	c.6531-12C>T		het	Alpha-Lely polymorphism	25%
P22	SPTA1	NM_003126	C.2898G>A	p.(=)	het	no	0	NA	NA	-29.3%	14.2	VUS
	SPTA1	NM_003126	c.6531-12C>T		het	Alpha-Lely polymorphism	25%
P23	ALAS2	NM_000032	c.-258C>G		het	Bekri et al	0.54%	NA	NA	NA		VUS
P23-1	ALAS2	NM_000032	c.-258C>G		absence
P24	TRPV4	NM_021625	c.1913C>T	p.P638L	hom	no	0.03% (no homozygotes)	B	DC	No effect		VUS
	ADAR	NM_001111	c.1586C>T	p.P529L	het	no	0	PD	DC	No effect		VUS
P25	SEC23B	NM_001172745	c.40C>T	p.R14W	het	Russo et al 2011	0.022%	PossD	DC	No effect		P
	SEC23B	NM_001172745	c.325G>A	p.E109K	het	Russo et al 2011	0.023%	PD	DC	No effect		P
P25-1	SEC23B	NM_001172745	c.40C>T	p.R14W	absence
	SEC23B	NM_001172745	c.325G>A	p.E109K	het
P25-2	SEC23B	NM_001172745	c.40C>T	p.R14W	het
	SEC23B	NM_001172745	c.325G>A	p.E109K	absence
*P26*	HAMP	NM_021175	c.49_54del	p.L17_L18del	het	no	0	NA	NA	No effect		VUS
	HFE	NM_000410	c.845G>A	p.C282Y	het		3.37%	PD	P	No effect		P
	CD46	NM_172359	c.402T>G	p.I134M	het	no	0	PossD	P	No effect		VUS
*P27*	CFH	NM_00186	c.2850G>T	p.Q950H	het	Mohlin et al 2015	0.39%	B	P	No effect		LB
*P27-1*	CFH	NM_00186	c.2850G>T	p.Q950H	Absence
*P27-2*	CFH	NM_00186	c.2850G>T	p.Q950H	absence
*P28*	SEC23B	NM_001172745	c.1276G>A	p.V426I	het	Schwartz et al 2009	4.3%	B	P	No effect		VUS
	CDAN1	NM_138477	c.256C>T	p.P86S	het	no	0.052%	B	P	No effect		VUS

*P29*	SPTA1	NM_003126	c.1688G>A	p.R563Q	het	no	0.11%	PD	DC	Possible new acceptor site	25.1	VUS
	HBB	NM_000518	c.20A>T	p.E7V	het	Yes coding for HbS	0.44%	B	P		13.8	P
	SPTA1	NM_003126	c.6531-12C>T		het	Alpha-Lely polymorphism
*P30*	PIEZO1	NM_001142864	c.1126C>G	p.P376A	het	no	0	B	P	Possible new acceptor site		VUS
*P31*	KCNN4	NM_002250	c.1055G>A	p.R352H	het	Rappetti Mauss et al 2015	0	PossD	DC	No effect		P
	PIEZO1	NM_001142864	c.3629C>T	p.A1210V	het	no	0.006%	B	DC	No effect		LP
*P31-1*	KCNN4	NM_002250	c.1055G>A	p.R352H	het	Rappetti Mauss et al 2015	0	PossD	DC	No effect		P
	PIEZO1	NM_001142864	c.3629C>T	p.A1210V	absence
*P32*	SPTB	NM_001024858	c.[6706C>A ;6737C>T]	p.[L2236M;A2246V]	Het in cis	no	0	B/PD	DC/P	No effect		VUS
	HBB	NM_000518	c.20A>T	p.E7V	het	Yes coding for HbS	0.44%	B	P		13.8	P
*P33*	G6PD	NM_000402	c.538G>A	p.V180I	het	no	0	PossD	DC	No effect		VUS
	SPTB	NM_001024858	c.6271C>A	p.P2091T	het	no	0.0065%	B	DC	no effect		VUS
*P33-1*	G6PD	NM_000402	c.538G>A	p.V180I	het
	SPTB	NM_001024858	c.6271C>A	p.P2091T	het
*P33-2*	G6PD	NM_000402	c.538G>A	p.V180I	absence
	SPTB	NM_001024858	c.6271C>A	p.P2091T	absence
*P34*	HFE	NM_000410	c.187C>G	p.H63D	hom	Kaczoeowska-Hac et al 2016	10.83%	B	P	No effect		LB
	ABCG8	NM_022437	c.-27G>A		het	no	0	NA	NA	NA		VUS
	ADAMTS13	NM_139025	c.119C>G	p.A40G	het	no	0.00041%	B	P			VUS
	ADAMTS13	NM_139025	c.4007G>A	p.R1336Q	het	no	0.0012%	PD	P			VUS
*P35*	SH2B3	NM_005475	c.1A>G	p.0?	het	no	0	NA	NA	No effect		LP
	SCN9A	NM_002977	c.2938G>T	p.A980S	het	no	0	PossD	DC	No effect		VUS
*P36*	SPTA1	NM_003126	c.6672A>C	p.E2224D	hom	no	1.5% no homozygotes	PD	DC	No effect	22.3	VUS
	SLC4A1	NM_000342	c.1199_1225del	p.A400_A408del	het	Wilder et al 2009	0.0047%	PD	DC			LP
	PIEZO1	NM_001142864	c.1369C>T	p.R457C	het	Russo et al 2018	0	PD	DC	No effect		LP
	G6PD	NM_000402	c.292G>A	p.V98M	het	Vulliamy et al 1988	1.15%					LP
	HBB	NM_000518	c.20A>T	p.E7V	het	Yes coding for HbS	0.44%	B	P		13.8	P
*P36-1*	SPTA1	NM_003126	c.6672A>C	p.E2224D	het
	SLC4A1	NM_000342	c.1199_1225del	p.A400_A408del	het
	PIEZO1	NM_001142864	c.1369C>T	p.R457C	het
	G6PD	NM_000402	c.292G>A	p.V98M	absence
	HBB	NM_000518	c.20A>T	p.E7V	het	Yes coding for HbS	0.44%	B	P		13.8	P
*P36-2*	SPTA1	NM_003126	c.6672A>C	p.E2224D	het
	SLC4A1	NM_000342	c.1199_1225del	p.A400_A408del	het
	PIEZO1	NM_001142864	c.1369C>T	p.R457C	het
	G6PD	NM_000402	c.292G>A	p.V98M	absence
	HBB	NM_000518	c.20A>T	p.E7V	het	Yes coding for HbS	0.44%	B	P		13.8	P
*P37*	SPTA1	NM_003126	c.3291G>A	p.W1097*	het	no	0	NA	NA	NA	42	LP
	SPTA1	NM_003126	c.6531-12C>T		het	Alpha-Lely polymorphism
	HFE	NM_000410	c.187C>G	p.H63D	hom	Kaczoeowska-Hac et al 2016	10.83%	B	P	No effect		LB
*P38*	CFH	NM_00186	c.157C>T	p.R53C	het	Servais et al 2012	0.0014%	PD	DC	No effect		LP
	PIEZO1	NM_001142864	c.4246G>A	p.G1416R	het	no	0.00033%	PD	DC	New cryptic acceptor site		VUS
*P39*	SPTA1	NM_003126	c.779T>C	p.L260P	het	Marchesi et al 1987	0.017% (Afr)	PD	DC	No effect		LP
	SPTA1	NM_003126	c.6531-12C>T		het	Alpha-Lely polymorphism
*P40*	ATP11C	NM_173694	c.2434C>T	p.P812S	hem	no	0.00055% no hemizygous	PD	DC	No effect		VUS
	ANK1	NM_020476	c.4558G>C	p.E1520Q	het	no	0.0021%	B	DC	No effect	26	VUS
*P40-1*	ATP11C	NM_173694	c.2434C>T	p.P812S	absence
	ANK1	NM_020476	c.4558G>C	p.E1520Q	Het
*P40-2*	ATP11C	NM_173694	c.2434C>T	p.P812S	het
	ANK1	NM_020476	c.4558G>C	p.E1520Q	absence

Variants description and classification according to ACMG guidelines as benign likely benign (LB), variant of uncertain significance (VUS), likely pathogenic (LP) or pathogenic (P). In silico study of missense variations was assessed thanks to Polyphen-2, Mutation taster andCADD score algorithm. HGMD professional and pubmed web interface were used to check for variants description in litterature. Abbreviations: het: heterozygous state; hom: homozygous state; hem: hemizygous state; F: female; M: male; HS: hereditary spherocytosis; gnomAD: genome agregation database https://gnomad.broadinstitute.org ; ND: not done; NA: not applicable; DC: disease causing; P: polymorphism; PD: probably damaging; PossD: possibly damaging; B: benign

UH GROUP

Twenty patients were classified as UH because of discordant or non-contributive clinical features and/or biological tests. Familial segregation study could be performed in 6 families (P25, P27, P31, P33, P36, P40, figure 2). In UH patients, NGS analysis allowed elucidation of the whole phenotype in 13 patients (P21, P22, P25, P29, P30, P31, P32, P35, P36, P37, P38, P39, and P40 = UH full phenotype elucidation sub-group). Phenotype could not be explained, totally or partially, in7 patients (P23, P24, P26, P27, P28, P33 and P34 = UH unsolved or partially solved sub-group).

In UH full phenotype elucidation sub-group, SPTA1 was the most frequently mutated gene (in 6 probands P21, P22, P29, P36, P37, and P39). Variations in SPTA1 result in HE, HPP or HS depending on variants type and phase. In P37 and P39, genotypes and phenotypic data (clinical features, blood smear) were suggestive of HPP. We could not clearly decipher between HE or HPP for P21, 22 and P29.

Other mutated genes in this fully elucidated sub-group, are PIEZO1 (P30 and P36, DHSt), KCNN4 (P31, GARDOS), SPTB (P32, elliptocytosis), SLC4A1 (P36, SEA ovalocytosis), CFH (P38, aHUS), ATP11C (P40), SH2B3 (P35, probable myelodysplasia) and SEC23B (P25, CDA2).

Those genes are principally encoding RBC membrane proteins. Among membrane genes, atypical cases have to be mentioned here. P30 had myelodysplasia and important hemolysis without any family history. She carried a constitutional (present in DNA from blood and hair bulb) heterozygous PIEZO1 variant of uncertain significance (VUS) (p.P376A) and had atypical ektacytometry results. P36 is one of the 3 A/S symptomatic patients explored in our study (P29, P32 and P36) an A/S woman originating from Comoros islands. She experienced spleen infarct after a long-distance flight and showed the association of a HbS trait, SEA ovalocytosis (SLC4A1 : p.Ala400_Ala408del), the G6PD MATERA A- p.V98M variant at heterozygous state [40], a homozygous SPTA1 variation (p.E2224D) and a PIEZO1 mutation (p.R457C) already involved in DHSt [16]. Her daughter (P36-1) also presented with hemolytic anemia and harbored the same mutations (the SPTA1 variant being heterozygous). Her blood smear showed anisopoikilocytosis and some stomatocytes. Another relative (P36-2) was explored and presented the same mutations as P36-1. He had an hemolytic anemia and a retinopathy typical of sickle cell disease.

Mutated genes different from membrane ones in the UH full phenotype elucidation sub-group are: SEC23B in P25 with CDA type 2 phenotype ; SH2B3 in P35 with a probable myelodysplasia ; CFH in P38 with aHUS; ATP11C in P40.P25 harbored 2 already described mutations in SEC23B gene at compound heterozygous state (familial segregation done, figure 2) allowing correction of diagnosis towards CDA type 2 and not HS as initially suspected. P35 has a heterozygous deleterious variation in SH2B3: c.1A>G (initiation codon loss) found thanks to exomic approach. This result allowed to reorientate diagnosis towards probable myeloproliferative neoplasm in this patient. SH2B3 somatic mutations have been reported in myeloproliferative neoplasms such as primary myelofibrosis [41]. He also carried a heterozygous VUS in the SCN9A gene c.2938G>T (p.A980S) which likely explained the severity of painful crises reported in this patient. SCN9A is involved in neurogenic painful syndromes [42]. P38 carried a heterozygous mutation in CFH (p.R53C), which had previously been found in patients with preeclampsia-related SHUa [43], in complement-related glomerulopathies [44] and in familial forms of AMD (age-related macular degeneration) [45]. She also carried a heterozygous PIEZO1 VUS (p.G1416R) but had normal ektacytometry and blood smear. In this case one single mutation probably explains the entire phenotype (AMD + preeclampsia + hemolysis + altered renal function). P40 carried a likely pathogenic hemizygous variation in ATP11C (p.P812S). This X chromosome variation is recorded in gnomAD in only one heterozygous female (no hemizygous males or homozygous females recorded). He also carried one VUS in ANK1. His blood smear was normal and ektacytometry showed atypical profile with only dehydration and no change in osmotic resistance Functional testing is in progress.

Association of variations in several CHA list genes has been found in 6 patients out of 13 of the UH full phenotype elucidation sub-group :

P29 HBB S mutation and SPTA1
P31, KCNN4 and PIEZO1
P32, HBB S mutation and SPTB
P36, HBB S mutation , SPTA1, SLC4A1, PIEZO1 and G6PD
P38, CFH and PIEZO1
P40, ATP11C and ANK1,

HFE gene has been explored only in case of iron overload association and only mentioned when clinically relevant genotype had been found. Five patients have 2 distinct altered genes and one has 5 distinct altered genes.

In UH unsolved or partially solved sub-group, P23 had a heterozygous ALAS2 promoter variation (c.-258C>G),which had previously been reported to cause X-linked sideroblastic anemia [46]. This variation is present in gnomAD at an allelic frequency of 0.54% with 39 hemizygous males, which suggests that it is most probably a rare benign polymorphism. No other relevant genetic variation was found. P27 is a child with major hemolytic anemia at birth and neonatal splenomegaly with thrombocytopenia. Her mother and maternal aunt and maternal grand-father had the same phenotype. She carried a heterozygous variation in CFH (p.Q950H) [47], which was absent in the mother and the maternal aunt and is therefore not responsible for CHA in this family disorder. No other relevant genetic variation was found. P28 had an atypical association of two heterozygous variants in two genes involved in CDA: one in SEC23B (p.V426I) and one in CDAN1 (p.P86S). No case of digenic inheritance has yet been reported in CDA. No other relevant genetic variation was found. Other patients only had a part of their phenotype explained by WES: P24, with TRPV4 variation possibly involved in osteonecrosis and a candidate ADAR variation for hemolysis ; P26, HAMP and HFE variations for iron overload, CD46 variation for susceptibility to hemolysis; P33, G6PD and SPTB variations, which combination cannot explain the severe phenotype ; P34, HFE, ABCG8, ADAMTS13 variations that could confer susceptibility to hemolysis and iron overload but do not explain the severity of phenotype. In P24, the candidate gene variations could be identified thanks to WES. A heterozygous missense variation was found in the gene encoding the RNA-specific Adenosine Deaminase (ADAR) (p.P529L). No ADAR mutation has been associated to date with congenital hemolysis in humans but several studies showed a crucial role in mouse erythropoiesis. This patient also carried an apparently homozygous variation of TRPV4 (p.P638L), present in the gnomAD database at an allelic frequency of 0.03% without any homozygotes recorded (over >138000 subjects tested). TRPV4 mutations have been found in skeletal dysplasia, arthropathies and in a familial form of osteonecrosis [48]. The TRPV4 mutation could explain osteonecrosis but not hemolysis. Other cases will not be developed further.

Association of CHA genes list variants had been found in 3/7 patients of this sub-group:

P28 : SEC23B and CDAN1
P33 : G6PD and SPTB
P34 : ADAMTS13 and ABCG8

In this study, exome sequencing allowed the identification of a genetic cause of CHA in 33 out of 40 patients (82.5%), including 20 HS patients (100%) and 13 UH patients (65%). All HS patients harbored mutations in already-known HS genes: ANK1, SLC4A1, SPTB, SPTA1. All identified variants (21, among which 13 novel) were classified as likely pathogenic or pathogenic loss-of-function mutations. A recent exomic study of 38 Chinese patients with suspected HS found mutations of ANK1 or SPTB in all patients [24]. Gallagher et al in 2019 [25] reported on a group of 24 recessive HS patients explored by WES and whole genome sequencing (WGS) all having mutations found in SPTA1. Our results, together with those already published, suggest that there are probably no other major HS genes. In mouse, beta-adducin deficiency has been shown to cause spherocytosis [49], but no mutations of adducins have yet been identified in human HS.

In UH patients, NGS analysis allowed diagnosis in 13 patients (P21, P25, P29, P31, P22, P30, P32, P35, P36, P37, P38, P39, and P40). Rate of positivity is lower in UH patients than in HS cases probably because of the lack of clear phenotype.

One important finding of our study is the frequent association of several genetic variants in different genes in a same patient, emphasizing the genetic complexity of CHA. Twenty two percents (9/40) of patients harbored several variations in different CHA genes. Among HS patients, only 3 (15%) had variations in other genes, whereas in the UH group, 9/20 (45%) had an association of several variants in different CHA genes and considering only full elucidation phenotype sub-group 6/13 (46%) presented a variants association. The presence of more genetic variants in single patients in the UH group suggests that this association probably contributes to the complexity of the phenotype. Phenotypical tests alone are not able to detect those types of variants association, which diagnosis is very important because of its therapeutical consequences as splenectomy. Such association of several deleterious variants in genes causing RBC diseases is poorly described [50-52] and is probably due to the ethnic origin, notably for P36 family, in coming from Comoros Islands, a region of endemic malaria. Plasmodium is known to exert a selective pressure on various RBC proteins [53].

Our study also emphasizes the usefulness of exomic approach in the field of CHA, both for discovery of candidate genes (as TRPV4 or ADAR) and diagnosis reorientation. Thanks to exomic capture, we reorientated initial diagnosis for P35 (probable myeloproliferative syndrome) and explained a part of the phenotype in P24, P26 and P33. The cases of P27 remained unsolved despite large familial investigations. Exomic capture allowed us to find a possible candidate cause for P24 osteonecrosis since this patient harbored a homozygous TRPV4 variation. TRPV4 encodes a non-selective calcium-permeant cation channel expressed in different cell types [54] but not in erythrocytes and it has never been associated with hemolysis. Mutations have already been involved in skeletal dysplasia, hereditary neuropathies, arthropathies [55] and recently in a familial case of osteonecrosis [48]. All variants reported are heterozygous and only one paper described a familial case of complex phenotype with severe intellectual disability and neuropathy associated with compound heterozygosity for TRPV4 mutations [56]. Our patient does not present any neurological symptoms. We also found an ADAR variation in this patient which could be a good candidate for hemolysis. No mutations in ADAR have yet been associated with hemolysis in human. Studies in mouse showed that ADAR was implicated in erythropoiesis [57;58]. ADAR heterozygous mutations are been found in dyschromatosis symmetrica hereditaria [59] and homozygous or compound heterozygous variations in Aicardi-Goutières syndrome [60].

During the last few years, several NGS studies of CHA have been published [14-24; 26-32; 61] and have shown their usefullness (Additional file1, Supplementary table S1). Two recent studies using targeted approaches [16;17] , focused on 21 index cases and 62 families respectively. They obtained a positive rate (elucidation of the genetic cause of anemia) of 62% and 65% of cases respectively. In our study, this rate was 82.5%. In the two former studies and ours, the positive rate appears to be considerably higher than the exome success rate obtained in other genetic diseases, such as cardiomyopathies or intellectual deficiencies: 62 to 82.5% for CHA versus 30% on average for cardiomyopathies or intellectual deficiencies [62;63]. The two recent studies of Wang R. et al 2018 and Gallagher et al 2019 concerning exome and CHA on large cohort [24;25] were only dedicated to HS patients. Our study is the biggest one with WES data in CHA patients. Regarding our results, exome sequencing in the field of CHA appears as highly performant, in HS but also in UH. Recommendations proposed by King et al in 2015 [13], and compiled by Kim et al in 2017 [64] concerning genetic testing for CHA diagnosis need to be updated, as they only recommended genetic testing in a limited number of cases as a second diagnostic step. A recent article of Rets et al in 2019 suggests that targeted NGS becomes the diagnosis standard tool in CHA molecular testing and that WES and WGS could represents the future [65]. Our results show that molecular diagnosis with WES could easily be democratized and is fundamental for understanding patients’ phenotype, adapting therapeutic approach (such as splenectomy) and permiting genetic counseling. Indeed, exomic approach appears to be very useful in UH cases and for complex phenotypes.

This work emphasizes the usefulness of exome sequencing in CHA to reach a right diagnosis for each patient. This allows clinical geneticists to provide a personally fitted genetic counseling and permits to specialized clinicians to adjust treatment in some cases When the first-line phenotypic analyses have not been successful in elucidating the disease cause, genetic tests using NGS and especially exome sequencing appears as very helpful to uncover the intricate genetic defects causing CHA and associated manifestations. The recent developments of exomic or genomic sequencing technologies make them the most suitable and cheapest approaches for the genetic diagnosis of these disorders, keeping in mind the absolute necessity of pluridisciplinar teams able to deal with incidental or unexpected findings if needed.

Democratization of NGS should lead to update the current recommendations and diagnosis strategy concerning the place of genetic testing in CHA. The frequent discovery of several mutations in different genes in a same patient also suggests to reconsider the genetic bases of CHA: apparently monogenic diseases may in fact be oligogenic, with satellite variations acting as modulators of the phenotype or resulting in new clinical entities.

Deeper mechanistic studies are warranted to better understand the relationship between genotype and phenotype, especially in patients with atypical or poorly described variants association and to explain VUS. However, some interactions may be challenging to investigate with currently existing tools. The development of next generation phenotypic tools, such as RBC metabolomics and proteomics, may represent new steps in the exploration of RBC genetic disorders and key tools for VUS exploration.

AMD: Age-Related Macular Degeneration AR: autosomic recessive; AD: autosomic dominant; CHA: congenital hemolytic anemia; DHSt: dehydrated hereditary stomatocytosis; GFF: glomerular filtration flow; HS: hereditary spherocytosis ; HPP: hereditary pyropoïkilocytosis; HE: hereditary elliptocytosis; MDS: myelodysplastic syndrome; RBC: red blood cell; MPE: membrane protein electrophoresis ; MPN: myeloproliferative neoplasms; NGS: next generation sequencing; TPI: triose phosphate isomerase; UH: unexplained hemolysis; aHUS: atypical hemolytic and uremic syndrome; VUS: variant of uncertain significance; WES: Whole exome sequencing; WGS: Whole genome sequencing; XLD: X-linked disorder

Ethics approval and consent to participate

All patients cited in the text have filled consent form for genetic analysis and consent to participate to this study.

Consent for publication

Consent for publication has been obtained.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request

Competing interests

No competing of interests

Funding

No funding

Authors' contributions

L.M.H. analyzed and validated genetic data and wrote the paper; A.A. developed and performed bioinformatic analyses and reviewed the paper; B.B. performed EMA tests, examinations of blood smears and reviewed the paper; M.S. was in charge of enzymatic activities measurement; C.G. and V.O. performed genetic tests; O.W.B. performed EMA tests and reviewed the paper; S.P. was responsible for sickle cell disease genetic exploration; V.P. performed ektacytometry analysis and reviewed the paper; K.G. made membrane proteins electrophoresis; M.B. made enzymatic activities measurement and designed panel genes list; C.G. Z.M., M.D., A.P., N.C.C. and M.M. provided patients for study; P.B. provided patients for study and technical support for research; S.M. supervised enzymatic activities measurement and hemoglobin studies; B.F. supervised the entire study, validated genetic data and wrote the paper; F.G. supervised the entire study, provided patients and reviewed the paper.

Acknowledgements

none

Disclosures of Conflicts of Interest

none

Gallagher PG. Diagnosis and management of rare congenital nonimmune hemolytic disease. ASH Educ Program Book. 12 mai 2015;2015(1):392‑9.
Gallagher PG. Hereditary elliptocytosis: spectrin and protein 4.1R. Semin Hematol. 1 avr 2004;41(2):142‑64.
Mohandas N, Gallagher PG. Red cell membrane: past, present, and future. Blood. 15 nov 2008;112(10):3939‑48.
Gallagher PG. Abnormalities of the Erythrocyte Membrane. Pediatr Clin North Am. déc 2013;60(6):1349‑62.
Andolfo I, Russo R, Gambale A, Iolascon A. New insights on hereditary erythrocyte membrane defects. Haematologica. 2016;101(11):1284‑94.
Perrotta S, Gallagher PG, Mohandas N. Hereditary spherocytosis. 2008;372:16.
Glele-Kakai C, Garbarz M, Lecomte MC, Leborgne S, Galand C, Bournier O, et al. Epidemiological studies of spectrin mutations related to hereditary elliptocytosis and spectrin polymorphisms in Benin. Br J Haematol. oct 1996;95(1):57‑66.
Picard V, Guitton C, Thuret I, Rose C, Bendelac L, Ghazal K, et al. Clinical and biological features in PIEZO1-hereditary xerocytosis and Gardos-channelopathy: A retrospective series of 126 patients. Haematologica. 24 janv 2019;haematol.2018.205328.
Prchal JT, Gregg XT. Red Cell Enzymes. ASH Educ Program Book. 1 janv 2005;2005(1):19‑23.
Bianchi P, Fermo E, Glader B, Kanno H, Agarwal A, Barcellini W, et al. Addressing the diagnostic gaps in pyruvate kinase deficiency: Consensus recommendations on the diagnosis of pyruvate kinase deficiency. Am J Hematol. janv 2019;94(1):149‑61.
Iolascon A, Esposito MR, Russo R. Clinical aspects and pathogenesis of congenital dyserythropoietic anemias: from morphology to molecular approach. Haematologica. 1 déc 2012;97(12):1786‑94.
Gambale A, Iolascon A, Andolfo I, Russo R. Diagnosis and management of congenital dyserythropoietic anemias. Expert Rev Hematol. mars 2016;9(3):283‑96.
King M-J, Garçon L, Hoyer JD, Iolascon A, Picard V, Stewart G, et al. ICSH guidelines for the laboratory diagnosis of nonimmune hereditary red cell membrane disorders. Int J Lab Hematol. 1 juin 2015;37(3):304‑25.
Hamada M, Doisaki S, Okuno Y, Muramatsu H, Hama A, Kawashima N, et al. Whole-exome analysis to detect congenital hemolytic anemia mimicking congenital dyserythropoietic anemia. Int J Hematol. sept 2018;108(3):306‑11.
Niss O, Chonat S, Dagaonkar N, Almansoori MO, Kerr K, Rogers ZR, et al. Genotype-phenotype correlations in hereditary elliptocytosis and hereditary pyropoikilocytosis. Blood Cells Mol Dis. oct 2016;61:4‑9.
Russo R, Andolfo I, Manna F, Gambale A, Marra R, Rosato BE, et al. Multi-gene panel testing improves diagnosis and management of patients with hereditary anemias. Am J Hematol. 1 mai 2018;93(5):672‑82.
Averbuch NS, Steinberg‐Shemer O, Dgany O, Krasnov T, Noy‐Lotan S, Yacobovich J, et al. Targeted next generation sequencing for the diagnosis of patients with rare congenital anemias. Eur J Haematol. 1 sept 2018;101(3):297‑304.
Barreto RDO, Arrizabalaga B, Hoz ABD la, García‐Orad Á, Tejada MI, Garcia‐Ruiz JC, et al. Detection of new pathogenic mutations in patients with congenital haemolytic anaemia using next-generation sequencing. Int J Lab Hematol. 1 déc 2016;38(6):629‑38.
Agarwal AM, Nussenzveig RH, Reading NS, Patel JL, Sangle N, Salama ME, et al. Clinical utility of next-generation sequencing in the diagnosis of hereditary haemolytic anaemias. Br J Haematol. 1 sept 2016;174(5):806‑14.
Roy NBA, Wilson EA, Henderson S, Wray K, Babbs C, Okoli S, et al. A novel 33-Gene targeted resequencing panel provides accurate, clinical-grade diagnosis and improves patient management for rare inherited anaemias. Br J Haematol. oct 2016;175(2):318‑30.
Kedar PS, Gupta V, Dongerdiye R, Chiddarwar A, Warang P, Madkaikar MR. Molecular diagnosis of unexplained haemolytic anaemia using targeted next-generation sequencing panel revealed (p.Ala337Thr) novel mutation in GPI gene in two Indian patients. J Clin Pathol. 18 oct 2018;jclinpath-2018-205420.
Moreno‐Carralero M-I, Horta‐Herrera S, Morado‐Arias M, Ricard‐Andrés M-P, Lemes‐Castellano A, Abio‐Calvete M, et al. Clinical and genetic features of congenital dyserythropoietic anemia (CDA). Eur J Haematol. 1 sept 2018;101(3):368‑78.
Koker SA, Karapinar T, Oymak Y, Bianchi P, Fermo E, Gozmen S, et al. Identification of a Novel Mutation in the SEC23B Gene Associated With Congenital Dyserythropoietic Anemia Type II Through the Use of Next-generation Sequencing Panel in an Undiagnosed Case of Nonimmune Hereditary Hemolytic Anemia. J Pediatr Hematol Oncol [Internet]. 1 oct 2018 [cité 6 mars 2019];40(7). Disponible sur: insights.ovid.com
Wang R, Yang S, Xu M, Huang J, Liu H, Gu W, et al. Exome sequencing confirms molecular diagnoses in 38 Chinese families with hereditary spherocytosis. Sci China Life Sci. 1 août 2018;61(8):947‑53.
Gallagher PG, Maksimova Y, Lezon-Geyda K, Newburger PE, Medeiros D, Hanson RD, et al. Aberrant splicing contributes to severe α-spectrin–linked congenital hemolytic anemia [Internet]. 2019 [cité 11 juin 2019]. Disponible sur: http://www.jci.org/articles/view/127195/pdf
Han JH, Kim S, Jang H, Kim SW, Lee MG, Koh H, et al. Identification of a Novel p.Q1772X ANK1 Mutation in a Korean Family with Hereditary Spherocytosis. Bandapalli OR, éditeur. PLOS ONE. 24 juin 2015;10(6):e0131251.
Lacy JN, Ulirsch JC, Grace RF, Towne MC, Hale J, Mohandas N, et al. Exome sequencing results in successful diagnosis and treatment of a severe congenital anemia. Mol Case Stud. juill 2016;2(4):a000885.
Khurana M, Edwards D, Rescorla F, Miller C, He Y, Sierra Potchanant E, et al. Whole-exome sequencing enables correct diagnosis and surgical management of rare inherited childhood anemia. Mol Case Stud. oct 2018;4(5):a003152.
Kim M, Park J, Lee J, Jang W, Chae H, Choi H, et al. Hemolytic anemia with null PKLR mutations identified using whole exome sequencing and cured by hematopoietic stem cell transplantation combined with splenectomy. Bone Marrow Transplant. déc 2016;51(12):1605‑8.
Errichiello E, Vetro A, Mina T, Wischmeijer A, Berrino E, Carella M, et al. Whole exome sequencing in the differential diagnosis of Diamond-Blackfan anemia: Clinical and molecular study of three patients with novel RPL5 and mosaic RPS19 mutations. Blood Cells Mol Dis. 1 mai 2017;64:38‑44.
Lin P-C, Chiou S-S, Lin C-Y, Wang S-C, Huang H-Y, Chang Y-S, et al. Whole-exome sequencing for the genetic diagnosis of congenital red blood cell membrane disorders in Taiwan. Clin Chim Acta [Internet]. oct 2018 [cité 18 oct 2018]; Disponible sur: https://linkinghub.elsevier.com/retrieve/pii/S0009898118305461
Ittiwut C, Natesirinilkul R, Tongprasert F, Sathitsamitphong L, Choed‐amphai C, Fanhchaksai K, et al. Novel mutations in SPTA1 and SPTB identified by whole exome sequencing in eight Thai families with hereditary pyropoikilocytosis presenting with severe fetal and neonatal anaemia. Br J Haematol [Internet]. [cité 6 mars 2019];0(0). Disponible sur: https://onlinelibrary.wiley.com/doi/abs/10.1111/bjh.15559
; on behalf of the ACMG Laboratory Quality Assurance Committee, Richards S, Aziz N, Bale S, Bick D, Das S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. mai 2015;17(5):405‑23.
Bartolucci P, Brugnara C, Teixeira-Pinto A, Pissard S, Moradkhani K, Jouault H, et al. Erythrocyte density in sickle cell syndromes is associated with specific clinical manifestations and hemolysis. Blood. 11 oct 2012;120(15):3136‑41.
King M-J, Telfer P, MacKinnon H, Langabeer L, McMahon C, Darbyshire P, et al. Using the eosin-5-maleimide binding test in the differential diagnosis of hereditary spherocytosis and hereditary pyropoikilocytosis. Cytometry B Clin Cytom. 74B(4):244‑50.
Girodon F, Garçon L, Bergoin E, Largier M, Delaunay J, Fénéant‐Thibault M, et al. Usefulness of the eosin-5′-maleimide cytometric method as a first-line screening test for the diagnosis of hereditary spherocytosis: comparison with ektacytometry and protein electrophoresis. Br J Haematol. 1 févr 2008;140(4):468‑70.
Dhermy D, Galand C, Bournier O, King M-J, Cynober T, Roberts I, et al. Coinheritance of α- and β-Spectrin Gene Mutations in a Case of Hereditary Elliptocytosis. Blood. 1 déc 1998;92(11):4481‑2.
Christensen RD, Nussenzveig RH, Reading NS, Agarwal AM, Prchal JT, Yaish HM. Variations in Both α-Spectrin (SPTA1) and β-Spectrin (SPTB) in a Neonate with Prolonged Jaundice in a Family where Nine Individuals Had Hereditary Elliptocytosis. Neonatology. 2014;105(1):1‑4.
Darbellay R, Mach‐Pascual S, Rose K, Graf J, Beris P. Haemoglobin Tunis-Bizerte: a new a globin 129 Leu–> Pro unstable variant with thalassaemic phenotype. Br J Haematol. 90(1):71‑6.
Vulliamy TJ, D’Urso M, Battistuzzi G, Estrada M, Foulkes NS, Martini G, et al. Diverse point mutations in the human glucose-6-phosphate dehydrogenase gene cause enzyme deficiency and mild or severe hemolytic anemia. Proc Natl Acad Sci. 1 juill 1988;85(14):5171‑5.
Oh ST, Simonds EF, Jones C, Hale MB, Goltsev Y, Gibbs KD, et al. Novel mutations in the inhibitory adaptor protein LNK drive JAK-STAT signaling in patients with myeloproliferative neoplasms. Blood. 12 août 2010;116(6):988‑92.
Fertleman CR, Baker MD, Parker KA, Moffatt S, Elmslie FV, Abrahamsen B, et al. SCN9A Mutations in Paroxysmal Extreme Pain Disorder: Allelic Variants Underlie Distinct Channel Defects and Phenotypes. Neuron. déc 2006;52(5):767‑74.
Fakhouri F, Roumenina L, Provot F, Sallée M, Caillard S, Couzi L, et al. Pregnancy-Associated Hemolytic Uremic Syndrome Revisited in the Era of Complement Gene Mutations. J Am Soc Nephrol JASN. mai 2010;21(5):859‑67.
Servais A, Noël L-H, Roumenina LT, Le Quintrec M, Ngo S, Dragon-Durey M-A, et al. Acquired and genetic complement abnormalities play a critical role in dense deposit disease and other C3 glomerulopathies. Kidney Int. août 2012;82(4):454‑64.
Yu Y, Triebwasser MP, Wong EKS, Schramm EC, Thomas B, Reynolds R, et al. Whole-exome sequencing identifies rare, functional CFH variants in families with macular degeneration. Hum Mol Genet. 1 oct 2014;23(19):5283‑93.
Bekri S. A promoter mutation in the erythroid-specific 5-aminolevulinate synthase (ALAS2) gene causes X-linked sideroblastic anemia. Blood. 13 mars 2003;102(2):698‑704.
Mohlin FC, Nilsson SC, Levart TK, Golubovic E, Rusai K, Müller-Sacherer T, et al. Functional characterization of two novel non-synonymous alterations in CD46 and a Q950H change in factor H found in atypical hemolytic uremic syndrome patients. Mol Immunol. juin 2015;65(2):367‑76.
Mah W, Sonkusare SK, Wang T, Azeddine B, Pupavac M, Carrot-Zhang J, et al. Gain-of-function mutation in TRPV4 identified in patients with osteonecrosis of the femoral head. J Med Genet. oct 2016;53(10):705‑9.
Gilligan DM, Lozovatsky L, Gwynn B, Brugnara C, Mohandas N, Peters LL. Targeted disruption of the ␤ adducin gene (Add2) causes red blood cell spherocytosis in mice. Proc Natl Acad Sci USA. 1999;6.
Nivaggioni V, Léonnet C, Ebbo M, Grados A, Schleinitz N, Picard V, et al. Multiple splenic infarctions in a malagasy patient whith both southeast asian ovalocytosis and a sickle cell trait. Hématologie. 20167-8;(4):284–287.
Favale F, Gardembas M, Pajot O, Saada V, Fénéant-Thibault M, Delaunay J, et al. Southeast Asian Ovalocytosis and A Sickle Cell Trait in a Young Patient with Sudden Retinal Stroke: A Fortuitous Association? Hemoglobin. déc 2009;33(6):475‑9.
Ravindranath Y, Goyette G, Johnson R. Southeast Asian ovalocytosis in an African-American family. Blood. 1 nov 1994;84:2823‑4.
Ma S, Cahalan S, LaMonte G, Grubaugh ND, Zeng W, Murthy SE, et al. Common PIEZO1 allele in African populations causes RBC dehydration and attenuates Plasmodium infection. Cell. 5 avr 2018;173(2):443-455.e12.
White JPM, Cibelli M, Urban L, Nilius B, McGeown JG, Nagy I. TRPV4: Molecular Conductor of a Diverse Orchestra. Physiol Rev. juill 2016;96(3):911‑73.
Nilius B, Voets T. The puzzle of TRPV4 channelopathies. EMBO Rep. févr 2013;14(2):152‑63.
Thibodeau ML, Peters CH, Townsend KN, Shen Y, Hendson G, Adam S, et al. Compound heterozygous TRPV4 mutations in two siblings with a complex phenotype including severe intellectual disability and neuropathy. Am J Med Genet A. 173(11):3087‑92.
Wang Q, Khillan J, Gadue P, Nishikura K. Requirement of the RNA Editing Deaminase ADAR1 Gene for Embryonic Erythropoiesis. Science. 1 déc 2000;290(5497):1765‑8.
Liddicoat BJ, Hartner JC, Piskol R, Ramaswami G, Chalk AM, Kingsley PD, et al. Adenosine-to-inosine RNA editing by ADAR1 is essential for normal murine erythropoiesis. Exp Hematol. oct 2016;44(10):947‑63.
Ansai O, Shigehara Y, Ito A, Abe R, Shimomura Y. A novel splice site mutation in the ADAR gene leading to exon skipping and dyschromatosis symmetrica hereditaria in a Japanese patient. Clin Exp Dermatol. déc 2016;41(8):933‑4.
Rice GI, Kitabayashi N, Barth M, Briggs TA, Burton ACE, Carpanelli ML, et al. Genetic, Phenotypic, and Interferon Biomarker Status in ADAR1-Related Neurological Disease. Neuropediatrics. juin 2017;48(3):166‑84.
Novel mutations in patients with hereditary red blood cell membrane disorders using next-generation sequencing [Internet]. [cité 20 nov 2018]. Disponible sur: https://reader.elsevier.com/reader/sd/pii/S0378111917305218?token=4FCE740DDB6CD8B16A6646FE87FCB3F1E4CAC04E041FE4BBA4A60E2CF2F5076B36CCEFCE8332CC8394A4CDC061B9ED6A
Seidelmann SB, Smith E, Subrahmanyan L, Dykas D, Ziki MDA, Azari B, et al. The Application of Whole Exome Sequencing in the Clinical Diagnosis and Management of Inherited Cardiovascular Diseases in Adults. Circ Cardiovasc Genet [Internet]. févr 2017 [cité 4 févr 2019];10(1). Disponible sur: https://www-ncbi-nlm-nih-gov.gate2.inist.fr/pmc/articles/PMC5245580/
Nambot S, Thevenon J, Kuentz P, Duffourd Y, Tisserant E, Bruel A-L, et al. Clinical whole-exome sequencing for the diagnosis of rare disorders with congenital anomalies and/or intellectual disability: substantial interest of prospective annual reanalysis. Genet Med Off J Am Coll Med Genet. 2018;20(6):645‑54.
Kim Y, Park J, Kim M. Diagnostic approaches for inherited hemolytic anemia in the genetic era. Blood Res. 2017;52(2):84.
Rets A, Clayton A.L, Christensen R.D, Agarwal A.M. Molecular diagnostic update in hereditary hemolytic anemia and neonatal hyperbilirubinemia. Int J Lab Hematol. 2019;41(Suppl. 1):95–101.

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Exome sequencing for diagnosis of congenital hemolytic anemia

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