Clinical and genomic heterogeneity of Diamond Blackfan anemia in the Russian Federation

Abstract

Background

Diamond Blackfan anemia (DBA) is a genetically and clinically heterogeneous ribosomopathy and inherited bone marrow failure syndrome characterized by anemia, reticulocytopenia and decreased erythroid precursors in the bone marrow with an increased risk of malignancy and in approximately 50%, physical abnormalities.

Methods

We retrospectively analyzed clinical data from 77 patients with DBA born in the Russian Federation from 1993–2014. In 74 families there was one clinically affected individual; in only three instances a multiplex family was identified. Genomic DNA from 57 DBA patients and their first-degree relatives was sequenced for mutations in RPS19, RPS10, RPS24, RPS26, RPS7, RPS17, RPL5, RPL11, RPL35a and GATA1.

Results

Severe anemia presented before 8 months of age in all 77 patients; before 2 months in 61 (78.2%); before 4 months in 71 (92.2%). Corticosteroid therapy was initiated after 1 year of age in the majority of patients. Most responded initially to steroids, while 5 responses were transient. Mutations in RP genes were detected in 35 of 57 patients studied: 15 in RPS19, 6 in RPL5, 3 in RPS7, 3 each in RPS10, RPS26 and RPL11 and 1 each in RPS24 and RPL35a; 24 of which having not been previously reported. In one patient a balanced chromosomal translocation involving RPS19 was found. No mutations in GATA1 were found.

Conclusion

In our cohort from an ethnically diverse population the distribution of mutations among RP genes was approximately the same as was reported by others, although within genotypes most of the mutations had not been previously reported.

Introduction

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized, in classical cases, by a moderate to severe hypoproliferative anemia that usually presents during infancy or early childhood [1]. Although red cell aplasia is the most prominent feature of DBA the disease is also characterized by growth retardation and congenital malformations, in particular craniofacial defects and defects of the upper limbs, heart, and genitourinary system present in ~30%–50% of patients [2–4] as well as a predisposition to cancer [5].

DBA is a clinically heterogeneous disease: laboratory findings, such as macrocytosis, elevated erythrocyte adenosine deaminase (eADA) activity and fetal hemoglobin (HbF) are observed in a majority of, but not in all, patients with DBA [6–8]. The incidence of DBA is estimated to be between 5–10 per million live births [2, 8–10]. It appears that approximately 40% of cases are familial, with disease inherited predominantly as an autosomal-dominant; the remaining cases are sporadic new dominants. The incidence is equal in both genders [8].

Boria et al. reviewed the molecular basis of Diamond Blackfan anemia, emphasizing that it is a disorder of defective ribosome biosynthesis [11]. DBA is genetically heterogeneous with confirmed mutations or deletions in 6 small subunit associated ribosomal protein genes (RP); RPS7, RPS10, RPS17, RPS19, RPS24, RPS26 and as well as 5 large subunit associated RPs; RPL5, RPL11, PRL19, RPL26 and RPL35a in approximately 65–70% of DBA patients [12], Other RP gene mutations, in RPS27, RPS28, RPS29 and RPL15, RPL19, RPL27, RPL31 are less firmly established [13–17] indicating that DBA is a disorder of ribosomal biogenesis and/or function [18–24]. Recently, additional kindreds have been identified harboring mutations in the erythroid transcription regulator GATA1 [25].

The aim of our study was to evaluate the presence of causative mutations in a genetically distinct and somewhat diverse cohort of patients from the Russian Federation and to compare our findings to those described in other cohorts.

Methods

Patients

We analyzed retrospective data from seventy-seven cases of DBA (41 male, 36 female) born in the Russian Federation over a 20-year period (1993–2014). The diagnosis of DBA in all probands and their family members was established according to the criteria of the DBA Working Group of the European Society for Paediatric Haematology and Immunology [2] based solely upon modified classical criteria; the presence of normochromic, often macrocytic, anemia; reticulocytopenia; a low number or lack of erythroid precursors in the bone marrow; a normal chromosome fragility test (diepoxybutane) and; in some patients, congenital malformations. Using these relatively stringent criteria, only three families were multiplex and seventy-four had only one clinically affected individual. Furthermore the ascertainment of only seventy-seven cases over a 20 year time period (3.85 cases/year) is below the number of cases expected [8] for the population size of the Russian Federation with approximately 1.9 million births/year suggesting incomplete case ascertainment.

Genetic studies

Genomic DNA was isolated from peripheral blood samples using the nucleic acid isolation kit AmpliPrime DNA-sorb B (Central Research Institute for Epidemiology, Moscow, Russia) according to the manufacturer's instructions. We amplified genomic DNA samples from fifty-seven unrelated DBA probands enrolled in the study and when available their first degree relatives by PCR and sequenced the products on an AB 3130xl Genetic Analyzer (Applied Biosystem, USA) for mutations in nine RP genes (RPS19, RPS10, RPS24, RPS26, RPS7, RPS17, RPL5, RPL11, RPL35a) and GATA1. All exons and exon-intron junction sites were studied. There was no ascertainment of copy number variants, thus it is probable that deletions of DBA-associated RP genes were undetected in this study (26). When sequence changes were found, independent PCR products were sequenced to confirm the mutations. In support that these sequence changes were not polymorphic variations we verified that none was reported in the Single Nucleotide Polymorphism database (dbSNP at www.nchi.nlm.nih.gov/SNP) or in the Ensembl database (www.ensembl.org).

Results

Severe normochromic anemia with low reticulocyte and normal platelet count manifested before the age of two months in sixty-one cases (78.2%). Twenty-eight of these cases presented at birth. Ten cases (12.8%) presented at the age of 3–4 months and six cases (7.7%) at the age of 5–8 months. One 3 month old female patient presented, in addition to anemia, with thrombocytopenia and granulocytopenia, both resolving spontaneously at 6 months of age. Congenital malformations, predominantly craniofacial defects and short stature, were observed in forty-six patients (59.1 %). Table 1 describes the spectrum of malformations observed in our patients. Per consensus guidelines (8) corticosteroids were withheld from the majority (N=56) of patients for up to or greater than 1 year of age with patients receiving regular red blood cell transfusions to keep their hemoglobin level in the 9.5–12.0 g/dL range. Steroid therapy was initiated at the age of 5–8 months in fourteen cases. Five of thirty-five patients who initially responded to steroid therapy had only a transient response. Two non-responders, male and female, achieved remission at the ages of 15 and 18 years, respectively. The female patient relapsed within 6 months. Two patients were successfully transplanted from an HLA-identical sibling at the ages of 1 and 2 years, respectively.

Table I.

Spectrum of physical malformations among 77 DBA patients

Malformation	Number of patients	%
Craniofacial dysmorphism*	23	29.9
Craniofacial dysmorphism, webbed neck	12	15.6
Craniofacial dysmorphism, cardiac malformation**	7	9.0
Craniofacial dysmorphism, cryptorchidism	1	1.3
Craniofacial dysmorphism, cardiac malformation, malformation of the gastrointestinal tract***, single kidney	2	2.6
Multiple malformation (craniofacial, heart, hypospadias, bone dysplasia, microcephaly, absent left ear)	1	1.3
None	31	40.3

^*Craniofacial defects observed in our cohort of patients includes micrognathia, high arched palate, ocular hypertelorism, epicanthus, broad flat nasal bridge, microtia, low-set ears and in two cases low anterior hairline.

^**Cardiac malformations included aortic septal defect, membranous subaortic stenosis, pulmonary stenosis, patent foramen ovale, ventricular septal defect, patent ductus arteriosus.

^***Malformation of the gastrointestinal tract – malrotation, megacolon.

At the time of the study twenty-two patients (33%) were steroid-dependent, three (4%) achieved complete hematologic recovery after long-term low-dose steroid therapy, two (3%) became transfusion-independent after bone marrow transplantation and forty patients (60%) still required regular transfusions.

Fifty-seven families were available for DNA analyses. Their data are shown in Table 2. Mutations in different RP genes were detected in thirty-five probands (15 in RPS19, 3 in RPS7, 3 in RPS26, 3 in RPS10, 1 in RPS24, 6 in RPL5, 3 in RPL11 and 1 in RPL35a). There were no patients with mutations in RPS17 or GATA1. Thirty-one cases among them appeared to be sporadic and only four were familial. This low number of familial cases is likely a consequence of incomplete genetic ascertainment and small family size. One patient (P2) showed a balanced chromosomal translocation involving RPS19 [t (8; 19) (q24; q13)]. Thirty-five percent of patients did not have any mutations in the studied genes.

Table II.

RP gene mutations, congenital anomalies and treatment response in DBA patients

Gene	Proband’s ID (gender), inheritance	Exon or intron	DNA mutation	Predicted amino acid change	Family member DNA results	Age of disease onset	Malformation status	Response at first steroid therapy/ age	Present therapy	Previously reported1
RPS19	P30(M) sporadic	Ex 2	c.3G>T	Met1>Ile	Parents not tested	1 mo	craniofacial dysmorphism*, webbed neck	Unresponsive at 12 mo	Trs (RBC transfusions)	Yes
RPS19	P74 (M) sporadic	Ex 2	c.3G>A	Met1>Ile	mother – normal, father not tested	At birth	None	Response at 18 mo, lost response at 38 mo	Trs	Yes
RPS19	P57(F) sporadic	Ex 2	c.29_30insA	Frameshift at codon 10; stop at 50 p.Asn10Lys_fs*41	Parents not tested	1 mo	craniofacial dysmorphism*	Steroid naive	Trs	No
RPS19	P60(M) sporadic	Ex 2	c.31C>T	Gln11*	Parents normal, de novo	At birth	craniofacial dysmorphism*	Unresponsive at 3 mo	Trs	Yes
RPS19	P25(M) sporadic	Ex 2	c.33delG	Frameshift at codon 11; stop at 28 p.Gln11His_fs*18	Parents normal, de novo	2 mo	craniofacial dysmorphism*, cardiomyopathy	Response at 4 mo	Steroid therapy	No
RPS19	P11 (F) sporadic	Ex 3	c.77_79del GGAinsTG	Frameshift at codon 27; stop at 28 p.Lys27Ser_fs*2	mother – normal, father not tested	1 mo	None	Response at 18 mo	Steroid therapy	No
RPS19	P24 (M) sporadic	Ex 3	c.114G>C	Lys38Asn	Parents normal, de novo	At birth	craniofacial dysmorphism*	Response at 3 mo	Steroid therapy	No
RPS19	P14 (F) sporadic	Ex 3	c.156G>A	Trp52*	Parents normal, de novo	At birth	None	Unresponsive at 23 mo	Trs	Yes
RPS19	P32(M) sporadic	Ex 4	c.184C>T	Arg62Trp	Parents not tested	2 mo	None	Unresponsive at 12 mo	Trs	Yes
RPS19	P22(M) sporadic	Ex 4	c.242_243 insA	Frameshift at codon 82; stop at 153 p.Arg82Thr_fs*72	Parents not tested	1 mo	craniofacial dysmorphism*, aortic septal defect, neutropenia	Response at 4 mo	Steroid therapy	No
RPS19	P17(M) sporadic	Ex 4	c.248_249 insA	Frameshift at codon 84; stop at 153 p.Arg84Glu_fs*70	Parents not tested	At birth	None	Response at 2 mo	Steroid therapy	No
RPS19	P16(M) sporadic	Int 4	c.356+1 G>C	Donor splice site mutation	Parents not tested	1 mo	None	Response at 18 mo	Steroid therapy	No
RPS19	P54(M) sporadic	Ex 5	c.386_387 delGA	Frameshift at codon 130; stop at 152 p.Asp130Ser_fs*23	Parents normal, de novo	1 mo	craniofacial dysmorphism*	Response at 5 y	Steroid therapy Remission at 20 years	No
RPS19	P62(M) sporadic	Ex 5	c.392T>G	Leu131Arg	mother normal, father not tested	At birth	High arched palate, ocular hypertelorism, microtia, low- set ears pulmonary stenosis, ventricular septal defect	Unresponsive at 6 mo	Trs	Yes
RPS19	P71(M) sporadic	Ex 5	c.406 G>T	Gly136*	Parents normal, de novo	At birth	None	Response at 26	Steroid therapy	No
RPS19	P2(M) sporadic	t(8;19)(q24;q13)c[13]			Parents normal	1 mo	craniofacial dysmorphism*, webbed neck	Response at 14 mo	Steroid therapy	No
RPS7	P8 (M) Familial	Int 1	c.-19+1 G>T	Donor splice site mutation	father and brother – same mutation, mother and grandparents - normal	8 mo	Craniofacial dysmorphism*	Response at 36 mo	Steroid therapy	No
RPS7	P18 (F) sporadic	Int 1	c.19+2T >C	Donor splice site mutation	Parents normal, de novo	1 mo	Craniofacial dysmorphism*	Response at 10 mo	Steroid therapy	No
RPS7	P75 (M) Familial	Int 2	c.75+2_75+8delTGAGAGG	Donor splice site mutation	father– same mutation, mother - normal	1 mo	None	Unresponsive at 36 mo	Trs	No
RPS10	P21 (M) sporadic	Ex 3	c.292_293insTACGGCC	Frameshift at codon 98; stop at 109 p.Arg98Leu_fs*12	Parents not tested	2 mo	Craniofacial dysmorphism*	Response at 24 mo	Trs at 7 y	No
RPS10	P67 (M) sporadic	Ex 4	c.337C>T	Arg113*	Parents normal, de novo	At birth	None	Response at 6 mo	Steroid therapy	Yes
RPS10	P64 (F) familial	Ex 4	c.337C>T	Arg113*	father – same mutation, mother - normal	At birth	None	Response at 10 mo	Steroid therapy	Yes
RPS24	P7 (F) sporadic	Int 1	c.3+1 G>T	Donor splice site mutation	Parents normal, de novo	8 mo	Craniofacial dysmorphism*	Unresponsive at 24 mo	Trs	No
RPS26	P1 (F) sporadic	Ex 2	c.8_11delAGAA	Frameshift at codon 4; stop at 43 p.Lys4Glu_fs*40	Parents not tested	3 mo	Craniofacial dysmorphism*	Response at 12 mo	Steroid therapy	No
RPS26	P3 (F) sporadic	Ex 2	c.28C>G	Arg10Gly	Parents normal, de novo	1 mo	Craniofacial dysmorphism*, ventricular septic defect, unfinished gut’s turn	Response at 6 mo	Steroid therapy	No
RPS26	P5 (M) sporadic	Int 3	c.312+3- c.312+6delGAGT	Donor splice site mutation	mother - normal, father not tested	2 mo	Craniofacial dysmorphism*, cryptorchidism	Unresponsive at 4 mo	Trs	No
RPL5	P77 (F) sporadic	Ex 2	c.47delA	Frameshift at codon 16; stop at 18	Parents not tested	At birth	None	Steroid naive	Trs	No
RPL5	P76 (F) sporadic	Ex 2	c.67C>T	Arg23*	Mother and sister – normal, father not tested	11 days	None	Response at 12	Steroid therapy	Yes
RPL5	P37 (M) sporadic	Ex 3	c.187C>T	Gln63*	Parents normal, de novo	1 mo	Craniofacial dysmorphism*, webbed neck	Unresponsive at 18 mo	Trs	No
RPL5	P4 (F) sporadic	Int 3	c.189+3insC	Donor splice site mutation	Parents normal, de novo	2 mo	Craniofacial dysmorphism*, low anterior hairline, webbed neck,	Unresponsive at 6 mo	Trs	No
RPL5	P66 (F) familial	Int 4 – Ex 5	c.325-4_c.332del ATAGCTTCTCAA insTT	Acceptor splice site mutation	mother and sister - normal, father – the same mutation	At birth	Craniofacial dysmorphism*	Unresponsive at 12 mo	Trs	No
RPL5	P40 (M) sporadic	Ex 5	c.351delG	Frameshift at codon 118; stop at 125 p.Ile118Ser_fs*8	mother - normal, father not tested	7 mo	Craniofacial dysmorphism*	Unresponsive at 24 mo	Trs	No
RPL11	P35 (M) sporadic	Ex 2	c.60_61delCT	Frameshift at codon 21; stop at 53	mother - normal, father not tested	1 mo	Craniofacial dysmorphism*	Unresponsive at 6 years	Trs	Yes
RPL11	P72 (F) sporadic	Ex 2	c.65delT	Frameshift at codon 22; stop at 33 p.Cys21Ser_fs*33	mother - normal, father not tested	1 mo	High arched palate, microtia, ocular hypertelorism, patent foramen ovale, patent ductus arteriosus	Steroid naive	Trs	No
RPL11	P73 (F) sporadic	Ex 3	c.223C>T	Arg75*	Parents not tested	At birth	High arched palate, microtia, ocular hypertelorism, patent foramen ovale,, duplicated left kidney	Steroid naive	Trs	Yes
RPL35a	P55 (F) sporadic	Ex 3	c.90delT	Frameshift at codon 30; stop at 74 p. Ile30Met_fs*45	Parents not tested	At birth	None	Response at 11 mo	Steroid therapy	No

¹http://www.dbagenes.unito.it/

^*Non-specific syndromic facies, not specified

A database of DBA-associated mutations, curated by Boria and Ramenghi, in the Leiden Open Variation Database (http://www.dbagenes.unito.it/) was used to ascertain the presence of novel mutations in known DBA-associated RP genes. The predominant mutations, as described in numerous cohorts were present in RPS19 (Table 2). Our patients were typical with regard to age at presentation, response to steroids and the nature of congenital anomalies; numbers being too small for a comparison to published data. Of note there were ten mutations previously not described and four known mutations associated with DBA. The mutations were predominantly frameshift mutations affecting the sequence of exons 2, 3, 4 and 5 (6 cases), five amino acid substitutions, three missense-mutations and one affecting the donor splice site of intron 4. In one case a balanced chromosomal translocation (t (8; 19) (q24; q13) c [13]) was found. De novo balanced reciprocal translocations have been described previously [27, 28].

Mutations in the RPS26 gene were identified in three probands. All of them were sporadic and were not described previously. Two mutations affected the sequence of the second exon (amino acid substitution (Arg10Gly) associated with multiple congenital malformations and a frameshift mutation with typical craniofacial dysmorphism). The two carrying these mutations responded to steroid therapy and remain dependent on low-dose corticosteroids. The third patient had a donor splice site mutation at the third intron associated with craniofacial dysmorphism and cryptorchidism. He remains transfusion-dependent.

Mutations in the RPL5 gene were identified in six probands. Five of them were sporadic, one familial. Five mutations were not described previously. These patients had craniofacial dysmorphism, webbed neck and poor hair growth in one case. All affected patients had their disease presentation during the first two months of life and were unresponsive to steroid therapy. Two probands were found to have a frameshift mutation. Two mutations affecting the splice sites were found: one (P4) was a de novo insertion near the donor splice site after exon 3 and the second one (P66) was acceptor splice site complex deletion-insertion mutation at the junction of intron 4 and exon 5 (c.325-4_c.332delATAGCTTCTCAAinsTT). This complex mutation was familial (father shares the same mutation, but he is not clinically affected). Two mutations were nonsense mutations at exon 2 and 3, respectively.

Two different mutations in RPS10 were identified in three unrelated probands. A nonsense mutation at exon 4 (Arg113Stop) was previously described [18]. This mutation was identified in two unrelated probands. Interestingly, one patient’s father bears the same nonsense mutation at exon 4 and has a MCV (99 fL) at the upper limit of normal. The other demonstrated this mutation de novo. In both cases, a response to initial steroid therapy was achieved. The other mutation is a new sporadic frameshift mutation at exon 3 associated with craniofacial dysmorphism. That patient initially responded to steroid therapy at 2 years of age but lost his response at 7 years of age and remains transfusion-dependent since then.

Three new mutations in the RPS7 gene were identified in three cases. All of them had not been described previously and affect the donor splice site at intron 1, one sporadic, and the other two familial. In all cases, these mutations were associated with craniofacial dysmorphism and response to initial steroid therapy. In one, at the age of 16.5 years, osteogenic sarcoma was diagnosed, representing the first case of DBA-associated osteogenic sarcoma associated with a mutation in RPS7. Following the initiation of cyclophosphamide chemotherapy the patient developed resistant aplasia and became transfusion-dependent for the remainder of his treatment. After discontinuation of chemotherapy, steroid therapy was initiated, which was successful, and his hemoglobin level and reticulocyte count were sustained on prednisolone (0.6 mg/kg/day). The patient’s affected brother and father shared the RPS7 mutation. The father had a low normal hemoglobin level (12 mg/dL) with a MCV (98 fL) at the upper limit of normal. The father’s parents did not share the mutation strongly suggesting that the father had a sporadic new dominant mutation.

The sporadic splice site mutation also affecting the donor site at intron 1 was detected in unrelated female proband (P18). Clinical manifestations appeared at the age of 1 month and until age 10 months she received regular blood transfusions. Subsequently, steroid therapy was initiated. The patient responded and remains on low dose prednisolone (0.5 mg/kg every other day).

An intronic deletion of seven nucleotides affecting the donor splice site of exon 2 was found in proband P75 who presented with mild anemia at birth and became transfusion dependent at the age of 2.5 years. His father bears the same mutation and has a MCV of 98 fL; his grandparents were not tested.

A mutation in the RPS24 gene was identified in one case. It had not been described previously. The patient presented with a normochromic are generative anemia at the age of 4 months. Steroid therapy was initiated twice at the age of 2 and 7 years old but was ineffective. The patient remained on regular packed red cell transfusions until at the age of 17 years. Spontaneous recovery occurred which lasted for 6 months, after which regular blood transfusions were again required. This patient carried a sporadic mutation - donor splicing site mutation at intron 1 – associated with craniofacial dysmorphism.

A previously described frameshift mutation in exon 2 of the RPL11 gene was identified in one patient and was associated with craniofacial dysmorphism and unresponsiveness to steroid therapy. Two of the other mutations (newly described frameshift mutation at exon 2 and a nonsense mutation at exon 3), previously described, were associated with craniofacial dysmorphism and cardiac malformation.

A novel frameshift mutation in exon 3 of the RPL35a gene was identified in one patient. No congenital malformations were observed in this patient. Initial steroid therapy was effective, and this patient continues to receive low-dose prednisolone (0.2 mg/kg/day).

Discussion

This report of patients with DBA from the Russian Federation is consistent with observations from previous reports and offers some new information describing novel mutations and a previously undescribed DBA-associated osteogenic sarcoma genotype (RPS7). Nonsense, splicing site and frameshift mutations all together were found in approximately 85% of cases, and missense mutations in 15% cases. We studied nine RP genes (RPS19, RPS10, RPS24, RPS26, RPL5, RPL11, RPL35a, RPS7 and RPS17) and found twenty five novel mutations in eight. Two mutations affecting the splice site after the first non-coding exon of the RPS7 gene were found to be pathogenic. One was found in two affected brothers and their father (mother and grandparents had normal DNA sequence of the RPS7 gene), the other appeared de novo (both parents being normal). This observation shows the importance of noncoding regions for ribosomal protein synthesis. The majority of our DBA cases were sporadic, as observed by Willig and colleagues (7) (10–25% of cases familial) and Gazda et al. (29) (40–50% of the cases familial) with autosomal dominant inheritance. Our four familial cases also demonstrated autosomal dominant inheritance. Twelve cases were sporadic with confirmed de novo mutations (both parents were studied). However the prevalence of familial cases is most certainly underestimated as a consequence of bias introduced in a retrospective study, the very restrictive definition of DBA employed and incomplete family data; only one parent, primarily the mother, was available for eight probands, and no genetic abnormalities were found in only that parent.

The mean age at disease presentation was 1 month, and approximately 69% of patients had associated physical anomalies, including craniofacial dysmorphism, webbed neck, atrial septal defect, patent foramen ovale, membranous subaortic stenosis, consistent with previous observations (reviewed in 30).

Also consistent with prior observations the majority of our DBA cases (16 probands) were associated with a mutation in the RPS19 gene and the frequency of other mutations in RP genes for our cohort of DBA patients is approximately the same as reported by others, save the Japanese registry which reports a RPS19 mutation frequency of only 11% (reviewed in 31).

Vlachos and colleagues (5) reported an increased risk of malignant disease in patients with DBA, with a likelihood of developing cancer of 22% by age 46 years. Among the tumors described osteogenic sarcoma was identified as a DBA-associated malignancy with an observed to expected ratio of 32.6. Unfortunately the paucity of genotypic data did not permit the identification of any osteogenic sarcoma associated mutations. Of note in the Russian DBA cohort, while only one patient developed osteosarcoma at age 17 years, a novel germ line mutation in RPS7 was demonstrated.

The Russian cohort demonstrates that additional important information can be garnered from the study of diverse international cohorts of patients with Diamond Blackfan anemia. It is of interest that the RP genes involved in DBA in Russia are proportionally similar to other cohorts, while the actual mutations vary significantly within each genotype.