EVALUATION OF GENETIC DISORDERS

All familial disorders are not necessarily genetically inherited (may be due to common environmental factors, e.g. malnutrition or infection) and similarly, all inherited disorders are not familial (may be due to new mutations).

Diagnostic evaluation for a genetic disorder is usually indicated on the basis of strong family history or clinical suspicion and aims to: (a) confirm the diagnosis in the index case, (b) detection of carrier state in parents/ siblings of index case; and (c) prenatal diagnosis in a suspected high-risk fetus.

Elaborate laboratory investigations are indicated only in selected cases with strong possibility of a known or unknown genetic defect.

This section deals with three important steps in genetic diagnosis: (a) suspecting the genetic disease, (b) clinical evaluation in these cases; and (c) laboratory workup in selected cases.

Step I. Suspecting the genetic disease: Family history and unexplained clinical features (Table 11.11) are most important clues to a genetic disease, although note that:

• A negative family history does not exclude genetic defect and the index case may be the only affected member of family, as recurrence risk is only 25-50% in single gene disorders and even lesser in multifactorial disorders or new mutations.

• A genetic defect may have variable expression, ranging from typical textbook description to very mild disease or physical stigmata.

• Similar phenotype may develop due to different genetic errors, e.g. arachnodactyly is seen in Marfan syndrome as well as homocystinuria.

TABLE 11.11: Indicators of genetic disease

a. Family history of...

- Similar disease with unexplained etiology

- Known genetic defect or carrier state

- Recurrent fetal/neonatal deaths

b. Clinical indicators (unexplained)

- Critically sick newborn/infant

- Failure to thrive

- Persistent vomiting

- Visceromegaly: Hepatomegaly, etc.

- Developmental delay/Intellectual disability

- Neurological: Seizures, coma, hypotonia, etc.

- Respiratory distress

- Hepatic: Icterus, Gallstones

- Hematological: Cytopenia, clotting defects

- Renal: Chronic kidney disease, stones

- Hearing/vision defects

- Dysmorphism: Abnormal skin, hair, eyes, genitals

- Skeletal dysplasia

c. Biochemical abnormalties (unexplained)

- Abnormal urinary odor/color

- Intractable acidosis/ketosis

- Hyperammonimia

- Hypoglycemia or dyselectrolytemia

• All genetic defects do not necessarily manifest at birth- some may present even beyond childhood or may not present at all due to variable penetrance.

Step II. Clinical evaluation: Clinical evaluation in a suspected genetic disorder aims to confirm the diagnosis or the need for further laboratory work-up. Some important aspects of clinical evaluation in these cases are as follows:

• Consanguinity, i.e. mating between close-relatives, is a major risk factor for unmasking of autosomal recessive defect with risk being directly proportional to strength of consanguinity, as follows:

- First-degree consanguinity, i.e. relationship between siblings or parent-offsprings.

- Second-degree consanguinity, i.e. relationship between uncle-niece, aunt-nephew or step-siblings.

- Third-degree consanguinity, i.e. relationship between first cousins.

• Pedigree charting: Pedigree charting is one of the most important tool to recognize genetic defects and their mode of inheritance, which requires careful history as well as examination of living relatives for at least three generations of index case (Proband or Propositus).

Common symbols used for pedigree construction are given in Fig. 11.9B. By convention, all members of same generation (living or dead) are placed in one horizontal line, with male members on the left side. Each generation is also denoted by roman numerals on left side. Fig. 11.9A has given a sample pedigree of an autosomal recessive disorders

• Constellation of clinical signs: Genetic frequently present with a constellation of clinical features, rather than an isolated abnormality.

Various databases like Online Mendelian Inheritance in Man (OMIM) or Phenomizer can be used to conduct search.

Step III. Laboratory evaluation: Laboratory evaluation of genetic disorders is often costly and indicated only in selected cases with strong possibility of a these defects, with choice depending on the probable clinical diagnosis.

Diagnostic investigations in genetic disorders may be broadly divided into (a) molecular genetic studies for suspected genetic disorders, and (b) Biochemical (enzymatic) studies for suspected inborn errors of metabolism. In addition, some overlapping cases may also need cytogenetic studies to rule out chromosomal disorders, discussed in Ch 11.3.

Fig. 11.9A and B: Pedigree charting: (A) Representative pedigree chart (B) with commonly used symbols.

TABLE 11.12: Suitable clinical handles (starting points) for genetic diagnosis

Dysmorphic features

• Stature: Tall/short/disproportionate stature

• Head: Microcephaly, Craniosynostosis

• Eyes: Cataracts, coloboma, retinitis

• Ears: Deafness, deformed ear, preauricular tags

• Mouth: Cleft lip/palate, large tongue

• Neck: Webbing, masses

• Limbs: Poly/syndactyly, dermatoglyphics

• Back: Spina bifida

• Genitals: Ambiguous genitals, micropenis

• Skin/hair: Dyspigmented skin patch/hair

Systemic abnormalities

• Organomegaly, e.g. hepatosplenomegaly

• Major malformations, e.g. CHD, gut atresias

• Skeletal abnormalities

Biochemical abnormalities

• Abnormal urinary odor

• Acidosis, hyperammonimia

• Recurrent/persistent icterus, hypoglycemia

Molecular genetic testing for single/multiple gene defects involve study of the specific DNA sequence from a sample of blood or other body fluids/tissues.

• Amplification methods to clone the sequence of interest in a DNA fragment using polymerase chain reaction (PCR) and form multiple copies, which can then be examined by specific probes or primers. Results are available within few hours and commonly used for genetic studies when very small quantities of DNA are available as well as to detect viral/bacterial DNA in infectious disorders.

Many variants of PCR tests are available at present including Real time PCR, Multiplex PCR, Nested PCR or Reverse transcriptase PCR ( RT-PCR), the last one to amplify RNA.

• Hybridization methods using DNA probes tagged with radioactive or fluorescent markers, to attach to the complementary sequence in subject's DNA. These hybridized probes can be later detected by various methods, e.g. FISH, MPLA, southern blotting, etc.

• Sequencing methods to compare the sequence of whole or part of the DNA from patient with reference population to detect genetic alterations and include two common methods - Sanger sequencing and Next generation sequencing (NGS).

Next generation sequencing (NGS): Only 1% of human DNA is used for protein coding (Exons), the rest being in non-coding regions (Introns), though it may regulate functions of Exons. NGS has emerged as the most powerful tool for diagnosis of monogenic disorders, which includes: (a) targeted panel testing of selected regions,

(b) exome sequencing of whole coding regions (ES), and

(c) whole genome sequencing (WGS) of coding as well as non-coding regions.

NGS involves fragmentation of the DNA sample into multiple fragments followed by capture of selected or entire DNA fragment for sequencing. This sequence,

when compared with reference population, may reveal many differences (variants), which may or may not be of pathogenic significance and need correlation with clinical phenotypes for interpretation.

NGS is usually preferred test in cases with overlapping or probably new phenotypes, for which precise genetic defect is difficult to specify with multiple possibilities. Biochemical studies are indirect but most frequently used methods to detect genetic disorders for which the actual enzymatic defect is known to cause the disease. These tests can be applied in the index case, prospective carrier or in the fetus for prenatal diagnosis and include:

(a) primary enzyme assays, e.g. hexosaminidase-A in Tay- Sachs disease; (b) non-enzymatic protein assays, e.g. fVIII assay for hemophilia; (c) secondary biochemical abnormality tests, e.g. CPK levels for Duchenne muscular dystrophy or Hb electrophoresis in hemoglobinopathies.

Many biochemical tests, though not diagnostic of a specific genetic disorder or IEM per se, help in short-listing the differential diagnosis in these defects, e.g. hyperammonemia, acidosis, hypoglycemia, etc. Relevance of these tests in diagnosis of IEM has been discussed in chapter of IEM.

11.5